Biomedical subjects

R Bassi

Publications and source records attributed to R Bassi.

At least 73 records · Page 4Linked to original sources

PubMed1997

Beta 1,4 N-acetylgalactosaminyltransferase (GM2/GD2/GA2 synthase) forms homodimers in the endoplasmic reticulum: a strategy to test for dimerization of Golgi membrane proteins.

Many Golgi membrane-bound glycosyltransferases exist as intermolecular disulfide bonded species, some of which have been demonstrated to be homodimers. Evidence for homodimer formation has come primarily from radiation inactivation experiments. We utilized an alternative strategy to test for homodimer formation of the cloned beta 1,4 N-acetylgalactosaminyltransferase (GalNAcT) responsible for synthesis of the glycosphingolipids GM2, GD2, and GA2. We stably transfected CHO cells with myc epitopetagged GalNAcT, which localizes primarily to the Golgi, and a hemagglutinin (HA) epitope-tagged GalNAcT fusion protein in which the cytoplasmic domain of GalNAcT was replaced by an ER retention signal. We then sought evidence for dimer formation between the two forms of GalNAcT. Immunoprecipitation with anti-myc or anti-HA co-immunoprecipitated the HA-tagged form or the myc-tagged form, respectively, providing evidence for the physical association of the two forms of GalNAcT. As a result of this association, GalNAcT/myc increased in the ER as demonstrated by Western blots and immunofluorescence. The rapid formation of dimers provided further evidence for dimer formation occurring in the ER. In summary, these results demonstrate that GalNAcT forms homodimers as a result of intermolecular disulfide bond formation in the ER. Furthermore, this ER motif strategy is potentially useful for demonstrating homodimer formation of other Golgi enzymes.

Animals↗

PubMed1997

Adult onset Krabbe's leukodystrophy: a report of 2 cases.

Krabbe's disease with adult onset is rare; neurological symptoms begin in childhood or at a juvenile age. Two brothers with adult onset of the disease are here reported; 1 sibling developed parapareto-ataxic gait while the other was asymptomatic. Magnetic resonance imaging showed areas of demyelination in the white matter of the brain, while nerve conduction was completely normal. In both patients deficiency of galactosylceramide beta-galactosidase was comparable to the infantile form.

Adult↗

PubMed1997

[The surgical treatment of hemorrhoids: diathermocoagulation and traditional technics. A prospective randomized study].

INTRODUCTION: The operations to treat hemorrhoids of III and IV degree are numerous but classifiable into two groups: "closed" techniques such as Ferguson, Parks and Khubchandani, and "open" techniques such as Arnous-Parnaud of the most famous Milligan-Morgan. Recently the Anglo-Saxon school was proposed a technical variant to Milligan-Morgan operation: it involves diathermy excision without peduncle ligature. This technique partly because of the coagulation of the pain receptor, partly because of the absence of "at risk" sutures in septic ground would involve a reduction in the postoperative pain. We have developed the following study to have a global valuation of these methodologies. MATERIALS AND METHODS: From November 1993, 135 patients with haemorrhoids of III and IV degree have been recruited and grouped in 3 random groups: the patients of the I group have been subjected to closed hemorrhoidectomy (Ferguson and is variant according to Khubchandani), the patients of the II group have been subjected to the Milligan-Morgan operation and those the III group of the diathermy hemorrhoidectomy. All the patients have had the same kind of preoperative preparation and the same kind of analgesic during the post-operative course. Before the operation all the patients were given a questionnaire including a scale of the pain on which the administrations of analgesic were also noted. RESULTS: A faster canalization and a minor postoperative pain were found in the patients of the II and III group and among them a minor use of analgesics in those subjected to diathermy-hemorrhoidectomy. No statistical difference occurred in postoperative haemorrhage into Milligan-Morgan and diathermy hemorrhoidectomy without peduncle ligature (2.2%). CONCLUSIONS: All the methods we have considered have turned out to be effective for the clinical and anatomical recovery. However, in our experience, the open techniques involve in a significant reduction of the postoperative pain and those made with diathermy permit a minor use of analgesics in comparison with those made a conventional scissor excision. Finally the peduncle ligature doesn't influence postoperative haemorrhage.

Chi-Square Distribution↗

PubMed1996

A CK2 site is reversibly phosphorylated in the photosystem II subunit CP29.

Protein phosphorylation is a major mechanism in the regulation of protein function. In chloroplast thylakoids several photosystem II subunits, including the major antenna light-harvesting complex II and several core complex components, are reversibly phosphorylated depending on the redox state of the electron carriers. A previously unknown reversible phosphorylation event has recently been described on the CP29 subunit which leads to conformational changes and protection from cold stress (Bergantino, E., Dainese, P., Cerovic, Z. Sechi, S. and Bassi, R. (1995) J. Biol Chem. 270, 8474-8481). In this study, we have identified the phosphorylation site on the N-terminal, stroma-exposed domain, showing that it is located in a sequence not homologous to the other members of the Lhc family. The phosphorylated sequence is unique in chloroplast membranes since it meets the requirements for CK2 (casein kinase II) kinases. The possibility that this phosphorylation is involved in a signal transduction pathway is discussed.

Amino Acid Sequence↗

PubMed1996

Beta1,4-N-acetylgalactosaminyltransferase (GM2 synthase) is released from Golgi membranes as a neuraminidase-sensitive, disulfide-bonded dimer by a cathepsin D-like protease.

Many Golgi membrane-bound glycosyltransferases are released from cells in a soluble form. To characterize this release process, we stably transfected Chinese hamster ovary cells with three myc epitope-tagged forms of cloned beta1, 4-N-acetylgalactosaminyltransferase (GalNAcT); two of these forms resided in the Golgi, while the third was retained in the ER. GalNAcT was released into the culture medium from cells transfected with the Golgi forms but not with the ER form of the enzyme. The medium from cells transfected with the Golgi forms contained disulfide-bonded dimers of GalNAcT, which carried neuraminidase sensitive, complex N-linked carbohydrate chains. This soluble species represented the major degradation product of cellular GalNAcT, which turned over with a half-time of about 1.7 h. The soluble species consisted of a mixture of truncated GalNAcT molecules, the major form of which was produced by cleavage near the boundary between the transmembrane and lumenal domains between Leu-23 and Tyr-24. This cleavage site fits the sequence pattern for sites cleaved by cathepsin D (van Noort, J.M., and van der Drift, A. C.M. (1989) J. Biol. Chem. 264, 14159-14164). These findings suggest that GalNAcT is converted from a membrane-bound to a soluble form as a result of cleavage by a cathepsin D-like protease in a compartment late in the Golgi secretory pathway.

Animals↗

PubMed1996

Conformational changes induced by phosphorylation in the CP29 subunit of photosystem II.

Light energy absorbed by the chloroplast membranes of higher plants is dissipated by nonradiative de-excitation in order to protect against photodamage. In photosystem II, which is the photosynthetic component most sensitive to photoinhibition, three pigment binding subunits, called CP24, CP26, and CP29, have been proposed to act in the regulation of the chlorophyll excited states concentration. In heavy stress conditions, CP29 becomes phosphorylated in its stroma-exposed portion, and this process is reversed by returning to normal conditions. In this study, we have used the pigments bound to the intramembrane hydrophobic portion of the protein as intrinsic probes to detect conformational changes induced by phosphorylation. We isolated the phosphorylated and unphosphorylated forms of the protein and showed that, although they have the same pigment complement, spectral differences can be consistently detected by absorption, linear dichroism, and circular dichroism spectroscopy. Alkaline phosphatase treatment of the phosphoprotein restores both the electrophoretic mobility and the spectral properties of the unphosphorylated CP29. The results of this study show that phosphorylation of CP29 can modulate the spectral properties of this photosystem II subunit and provide a possible mechanism for the regulation of excitation energy supply to the reaction center.

Alkaline Phosphatase↗

PubMed1996

Salvage of catabolic products in ganglioside metabolism: a study on rat cerebellar granule cells in culture.

Cerebellar granule cells in culture were subjected to a pulse (0.5-4 h)-chase (0-4 h) of 10(-6) M [3H]ganglioside GM1 carrying the radioactive label at the level of NeuAc ([3H-NeuAc]GM1), Sph ([3H-Sph]GM1) or Gal ([3H-Gal]GM1) and the formed [3H]metabolites were determined. With all forms of [3H]GM1, there was formation of [3H]catabolites, including [3H]H2O and [3H]biosynthetic products obtained by recycling of [3H]NeuAc, [3H]Sph and [3H]Gal released during intralysosomal ganglioside degradation (salvage processes). Much higher amounts of [3H]H2O were produced from [3H-Gal]GM1 than [3H-Sph]GM1 and [3H-NeuAc]GM1; conversely, more products from salvage processes (polysialogangliosides GD1a, GD1b, GT1b, O-acetylated GT1b, protein-bound radioactivity) were obtained with [3H-NeuAc]GM1 than the two other forms of [3H]GM1. Liberated [3H]NeuAc produced 10-fold less tritiated water and 10-fold higher salvage products than [3H]Gal. Using [3H-NeuAc]GM1, granule cells appeared to metabolize 7.7% of membrane-incorporated exogenous GM1 per hour with a high degree of NeuAc recycling and the calculated metabolic half-life was 6.5 h.

Animals↗

PubMed1996

Excited state equilibration in the photosystem I-light-harvesting I complex: P700 is almost isoenergetic with its antenna.

Photosystem I with its full antenna complement (PSI-LHCI) has been prepared by mild detergent solubilization with octyl beta-D-glucopyranoside from maize thylakoids. A preliminary polypeptide analysis is presented. At room temperature, the steady-state fluorescence derives from an almost perfectly thermalized state, as demonstrated by a Stepanov analysis, in which about 90% of the excited states are associated with the red chlorophyll spectral forms absorbing above 700 nm. Equilibration is temperature-sensitive and is lost at T < 200 K. A careful analysis of fluorescence between 75 and 280 K clearly demonstrates the presence of at least three red chlorophyll spectral forms with emission maxima at 720, 730, and 742 nm, the absorption origin bands of which have been calculated at 714, 725, and 738 nm. On the basis of a minor deviation from thermal equilibration around 695 nm, it is suggested that at least 3-4 antenna chlorophylls, with an average absorption near 695 nm, are strongly coupled to P700. Thermodynamic analysis of absorption and fluorescence spectra indicates that the equilibrium, absorption-weighted excited state population of the P700 dimer is around 0.013 assuming that the low-energy exciton state possesses all the oscillator strength. The average free energy for excitation transfer from antenna to P700 is thus calculated to be -0.26 kT at room temperature. This indicates that P700 is almost isoenergetic with its antenna at room temperature when the red forms are taken fully into account. From the calculated excited state population of P700, we estimate that the primary charge separation rate in PSI is 1-2 ps-1.

Light-Harvesting Protein Complexes↗

PubMed1996

Reconstitution and pigment-binding properties of recombinant CP29.

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.

Apoproteins↗

PubMed1996

Sphingoid bioregulators in the differentiation of cells of neural origin.

The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [3H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C2-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation.

Animals↗

PubMed1996

Cryoglobulinemic neuropathy related to hepatitis C virus infection. Clinical, laboratory and neurophysiological study.

Peripheral neuropathy is frequently reported in mixed cryoglobulinemia. As a close relationship has been established between mixed cryoglobulinemia and hepatitis C virus (HCV), the clinical, neurophysiological, and serologic findings of five patients affected by neuropathy, cryoglobulinemia and HCV infection were investigated. HCV infection was ascertained by the presence in the serum of anti-HCV antibodies detected by enzyme-linked immunosorbent assay(ELISA), and of HCV RNA assessed by polymerase chain reaction (PCR). Initial symptoms included paresthesias and painful dysesthesias in the legs. Later on, the patients developed a mainly asymmetric axonal polyneuropathy or a multifocal neuropathy associated with signs of systemic vasculitis. In this case series we report the short-term prognosis, as well as the response to interferon (IFN) alpha and conventional treatment. The presence of HCV RNA supports the hypothesis that a persistent HCV infection might be involved in the pathogenesis of mixed cryoglobulinemia and cryoglobulinemia-associated disorders.

Adult↗

PubMed1995

A mediator role of ceramide in the regulation of neuroblastoma Neuro2a cell differentiation.

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [3H]sphingomyelin or [3H]sphingosine or L-[3H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, beta-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

Animals↗

PubMed1995

Biochemical and functional properties of photosystem II in agranal membranes from maize mesophyll and bundle sheath chloroplasts.

We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non-denaturing lauryl beta-D- iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light-harvesting complex II (LHCII) that was somewhat over-represented, while the minor antenna complexes CP26 and CP29 were under-represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33-kDa protein of the oxygen-evolving enhancer polypeptides was not detectable nor was the ferredoxin-NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect of the QA- reoxidation. An additional specificity of these centers is the slow rate (50-ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1-2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow beta-phase of the induction kinetics.

Chlorophyll↗

PubMed1995

A post-translational modification of the photosystem II subunit CP29 protects maize from cold stress.

The resistance of maize plants to cold stress has been associated with the appearance of a new chlorophyll a/b binding protein in the thylakoid membrane following chilling treatment in the light. The cold-induced protein has been isolated, characterized by amino acid sequencing, and pulse labeled with radioactive precursors, showing that it is the product of post-translational modification by phosphorylation of the minor chlorophyll a/b protein CP29 rather than the product of a cold-regulated gene or an unprocessed CP29 precursor. We show here that the CP29 kinase activity displays unique characteristics differing from previously described thylakoid kinases and is regulated by the redox state of a quinonic site. Finally, we show that maize plants unable to perform phosphorylation have enhanced sensitivity to cold-induced photoinhibition.

Amino Acid Sequence↗

PubMed1995

The degradative pathway of gangliosides GM1 and GM2 in Neuro2a cells by sialidase.

Gangliosides GM1 [3H-labeled at the sphingosine (Sph) moiety] and GM2 [3H-labeled at the Sph or N-acetylgalactosamine (GalNAc) moiety] were administered to cultured Neuro2a cells for varying pulse (1-4 h) and chase (up to 4 h) periods, and their metabolic processing was followed. The main and earliest formed 3H-metabolites of [Sph-3H]GM1 were GM2, asialo-GM1 asialo-GM2, and lactose-ceramide, and those of [Sph-3H]GM2 were asialo-GM2 and lactose-ceramide. The asialo-GM1 and asialo-GM2 formed were isolated and chemically characterized. [3H]Asialo-GM2 was produced in identical amounts after treatment with equimolar [Sph-3H]GM2 and [GalNAc-3H]GM2. At low temperature or in the presence of chloroquine, the formation of all 3H-metabolites, including asialo-GM2 and asialo-GM1, was undetectable, indicating that ganglioside metabolic processing was an endocytosis- and lysosome-dependent process. These results demonstrate that in Neuro2a cells exogenous GM1 (and GM2) is mainly degraded through the pathway GM1-->GM2-->asialo-GM2-->-->Sph, with a minor fraction of GM1 undergoing degradation with the sequence GM1-->asialo-GM-1-->asialo-GM2-->-->Sph. These findings are consistent with the hypothesis that Neuro2a cells contain a sialidase (likely of lysosomal nature) affecting ganglioside GM1 and GM2. The sialidase-mediated degradative pathway of GM1 and GM2 in Neuro2a cells might be related to the tumoral nature of these cells.

Animals↗

PubMed1995

Personality disorders and psychiatric symptoms in psoriasis.

Two groups of psoriatic outpatients (ns = 192 and 119) were given, respectively, the Million Clinical Multiaxial Inventory-II and Foulds' Delusions-Symptoms-States Inventory. They were compared with dental (n = 192) and with general surgical (n = 190) patients. The psoriatic group presented clearly higher mean scores and frequencies on most of the personality disorder scales. On Foulds' inventory, psoriatic patients showed higher frequencies of neurotic and psychotic class allocations. A cluster analysis of personality scores provided evidence for 4 different personality clusters of patients with psoriasis: (a) Avoidant, Dependent, Schizoid, and Self-defeating (32.2%), (b) Compulsive, Narcissistic, and Aggressive (30.7%), (c) no personality disorder (18.2%), (d) Borderline, Paranoid, and Schizotypal, etc. (18.8%).

Adolescent↗

PubMed1994

Formation of bioactive sphingoid molecules from exogenous sphingomyelin in primary cultures of neurons and astrocytes.

Exogenous sphingomyelin, radiolabelled at the sphingosine moiety, was administered to primary cultures of cerebellar granule cells and astrocytes for different pulse times (20 min-2 h) and the fate of the radioactivity was followed. Ceramide was the main metabolic product in both cells, whereas sphingosine, glucosyl-ceramide and gangliosides GM3 and GD3 were produced only in astrocytes. When endocytosis was prevented and the lysosomal apparatus inactivated, ceramide formation was reduced slightly in granule cells and almost completely blocked in astrocytes, with disappearance of sphingosine, glucosyl-ceramide, GM3 and GD3. These data indicate that (a) ceramide is rapidly produced in cerebellar granule cells and astrocytes, presumably at the level of the plasma membrane in the first cell type, and of the lysosomes in the second one; (b) sphingosine is produced in cerebellar astrocytes by lysosomal sphingomyelin degradation and is partly reused for glucosyl-ceramide and ganglioside biosynthesis.

Animals↗

PubMed1994

Cultured cerebellar granule cells, but not astrocytes, produce an ester of ganglioside GD1b, presumably GD1b monolactone, from exogenous GD1b.

Granule cells and astrocytes from rat cerebellum were fed in culture with 2 microM ganglioside [Gal-3H]GD1b and then analysed for the presence of carboxyl esters of that ganglioside. Before extraction and purification of gangliosides, cells were treated with NaBH4 under conditions that would allow complete reductive cleavage of carboxyl ester linkages, [Gal-3H]GD1b monolactone and dilactone being used as reference esters of GD1b. These conditions, established by adding harvested cells (250 micrograms of protein) with 0.01-2 nmol of standard [Gal-3H]GD1b monolactone or dilactone and [Gal-3H]GD1b-1ol or -2ol formed respectively, consisted of an NaBH4/cell protein ratio of 2:1 (w/w). Cerebellar granule cells, but not astrocytes, were able to produce a radioactive compound which was identified as GD1b-1ol. The formation of this compound increased with pulse (up to 4 h) and chase (up to 3 h) time after a 2 h pulse and also occurred when ganglioside endocytosis was blocked. It can be concluded that cerebellar granule cells are able to convert ganglioside GD1b into a carboxyl ester form, presumably GD1b monolactone. The natural occurrence of the same GD1b carboxyl ester in cerebellar granule cells was also demonstrated.

Animals↗